1. Field of the Invention
The invention relates to continuous cell lines derived from normal adult human liver tissue. These cell lines display morphological and gene expression characteristics consistent with parentage of normal human hepatocytes. The cell lines are immortalized by expression of the large T antigen (TAg) of the SV40 virus, but are not tumorigenic. As such, they provide a reproducible source of cells for studies of initiation and progression of carcinogenesis, especially chemical and viral carcinogenesis caused by liver metabolism of non-tumorigenic precursor compounds to genotoxic substances or by infection by oncogenic hepatitis viruses.
2. Description of the Related Art
Throughout this patent application, numerous articles of the scientific literature are cited. Each of these references is hereby incorporated in its entirety by such citation.
Kaighn and Prince (1) described clonally-derived cultures of liver cells from fetal, infant and adult human donors more than 20 years ago. These cultures all had limited lifespans. Their observations suggested the existence of normal adult human liver epithelial cells that are either less differentiated, or are capable of undergoing retrograde differentiation into a form that is capable of completing a few population doublings in vitro if cultured under appropriate conditions. Cultures of rat liver epithelial cells have been established (2,3), but these cultures, like those of Kaighn and Prince, have only limited life span. Recently, we described a serum-free culture medium (LCM, described below) which supports extended replication of normal human liver epithelial cells (4). However, the growth potential of liver cells on this medium was still limited, that is, no more than 12 rounds of cell division were ever obtained in any of our cultures. All of these cultures are not suitable for long-term studies due to their limited lifespan.
Metabolic activation of environmental carcinogens from several chemical classes have been studied in human liver tissue explants or microsomes and isolated human hepatocytes. (5). Furthermore, observed animal species-specific differences in aflatoxin B.sub.1 (AFB.sub.1) and 2-acetylaminofluorene metabolism indicate the need for studying human liver or hepatocytes. However, because tissue availability is limited, individuals vary in their propensity for xenobiotic metabolism and reproducible in vitro conditions are difficult to establish, a reproducible system with human liver cells for pharmaco-toxicological studies has not been established.
Several groups of investigators have reported that the longevity of cultures of human epithelial cells can be increased or in some cases made indefinite (5,6) by transformation with the SV40 virus large T antigen (TAg) gene. Such transformed cells may have near normal karyotype and some of the isolates retain many of the growth and differentiation characteristics of their normal counterparts, including non-tumorigenicity. In addition, Woodworth et al. and Ledley et al. (7-9) have reported that rat hepatocytes transformed with an SV40 TAg gene retained several normal hepatocyte characteristics. Unfortunately, such cultures are not useful for studies of human carcinogenesis and drug metabolism studies, since metabolism of xenobiotic compounds can be very different in humans and in rats.